Am J Physiol Heart Circ Physiol 285: H26–H37, 2003.
First published March 13, 2003; 10.1152/ajpheart.00788.2002.
Conducted dilations initiated by purines in arterioles
are endothelium dependent and require endothelial Ca2⫹
Tasmia Duza1 and Ingrid H. Sarelius2
1
Department of Biomedical Engineering and 2Department of Pharmacology
and Physiology, University of Rochester, Rochester, New York 14642
Submitted 6 September 2002; accepted in final form 11 March 2003
conducted response; endothelium-dependent dilation; microvascular communication
axially along the
blood vessel wall is one phenomenon by which stimuli
sensed by a localized region of the vasculature are
communicated to remote regions (typically defined as
⬎1,000 ␮m upstream). Local and remote changes in
resistance acting in concert match the supply of oxygen
and other nutrients to localized metabolic needs. The
ultimate vasomotor outcomes of axially communicated
signals are often referred to as conducted responses
and have been observed in response to a wide range of
vasoactive molecules (15, 18, 27). ATP is one such
metabolically related nucleotide (22) that has also been
implicated as an autocrine and paracrine signaling
molecule in numerous cell systems (13, 29).
The vascular wall is decorated with purinergic receptors, which are divided into two main families. P1 purinergic receptors are defined as preferentially binding
adenosine (Ado) ⬎ AMP ⬎ ADP ⬎ ATP, whereas the
reverse is defined for P2 purinergic receptors (5). Purines
play an essential role in the regulation of vascular resistance. Stimulation of P1 receptors by Ado is a well-established dilator pathway, although the relative importance
of endothelial cells (ECs) versus smooth muscle cells
(SMC) is still unclear (31). In contrast, a clear understanding of the function of P2 receptors and the effects of
ATP in the microvasculature remains largely undefined.
One reason for this is that ATP can have either vasoconstrictor or vasodilator effects on arterioles (6). The presence of ecto-ATPases, which rapidly degrade ATP, generally maintains ATP between nanomolar and micromolar concentrations in the extracellular space (13, 29).
Appreciable levels of ATP can however occur transiently
(3, 6, 16) and may underlie one of the mechanisms by
which blood flow is regulated.
The goal of this study was to characterize the signaling pathway underlying the local and conducted (upstream) vasomotor response of intact blood-perfused
arterioles to ATP. Using the terminal vasculature of
hamster cheek pouch as a model, we identified the
primary cell type targeted by extracellular purines. We
also investigated the importance of P2 versus P1 purinergic receptors and the role of EC Ca2⫹ as a second
messenger molecule for both local and conducted dilations.
THE TRANSMISSION OF VASOMOTOR SIGNALS
Address for reprint requests and other correspondence: I. H. Sarelius,
Dept. of Pharmacology and Physiology, Univ. of Rochester Medical
Center, Box 711, Rochester, NY 14642 (E-mail: ingrid_sarelius
@urmc.rochester.edu).
H26
METHODS
General Methods
All protocols were approved by the Animal Care and Use
Committee of the University of Rochester and performed in
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
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Duza, Tasmia, and Ingrid H. Sarelius. Conducted dilations initiated by purines in arterioles are endothelium dependent and require endothelial Ca2⫹. Am J Physiol Heart
Circ Physiol 285: H26–H37, 2003. First published March 13,
2003; 10.1152/ajpheart.00788.2002.—The signaling pathways underlying the regulation of vascular resistance by
purines in intact microvessels and particularly in communication of remote vasomotor responses are unclear. One process by which remote regions of arterioles communicate is via
transmission of signals axially along the vessel wall. In this
study, we identified a pathway for local and conducted dilations initiated by purines. Adenosine (Ado) or ATP (bind P1
and P2 purinergic receptors, respectively) was micropipette
applied to arterioles (maximum diameter ⬃40 ␮m) in the
cheek pouch of anesthetized hamsters. Observations were
made at the site of stimulation (local) or ⬃1,200 ␮m upstream along the same vessel. P2 antagonists (pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid tetrasodium and
suramin) inhibited local constriction to ATP, whereas local
and upstream dilations were unaffected. In contrast, during
inhibition of P1 receptors (with xanthine amine congener) the
local constriction was unchanged, whereas both local and
upstream dilations to ATP were inhibited. Hydrolysis of ATP
to Ado is implicated in the dilator response as blocking
5⬘-ectonucleotidase (with ␣,␤-methyleneadenosine 5⬘-diphosphate) attenuated ATP-induced dilations. After endothelium
denudation, constriction to ATP was unchanged, but dilations to both ATP and Ado were inhibited, identifying endothelial cells (ECs) as the primary target for P1-mediated
dilation. Purines increased EC Ca2⫹ locally and upstream.
Chelation of EC Ca2⫹ (with BAPTA) abolished the local and
upstream dilations to P1 receptor stimulation. Collectively,
these data demonstrate that stimulation of P1 receptors on
ECs produces a vasodilation that spreads to remote regions.
There is an associated increase in EC Ca2⫹, which is a
required signaling intermediate in the manifestation of both
the local and axially communicated arteriolar dilations.
ARTERIOLAR DILATIONS INITIATED BY PURINES
ing a 1-min baseline period, a 2-min agonist application
period, and a 3-min recovery period in every experiment
(standard observation protocol) at either the agonist application site (local) or a site ⬃1,200 ␮m upstream (upstream site)
along the same vessel. A schematic of the experimental site is
shown in Fig. 1. Diameter measurements were reproducible
to ⫾0.5 ␮m. To determine the distance from the local site
(measured from the tip of the agonist pipette) to the upstream site, the vessel was traced in sequential fields of view
using a ⫻10 objective (numerical aperture 0.22) and recorded
on videotape. Distance measurements were made offline and
were accurate to ⫾30 ␮m.
The vascular responsiveness of each preparation was evaluated at the end of all experimental protocols. Only data
collected on preparations that displayed constriction to 10%
O2 and dilation to 10⫺4 M ACh or 10⫺3 M sodium nitroprusside (SNP) were kept for analysis (⬃5% of all preparations
were discarded). Vessel diameter following at least 3 min of
superfusion of the entire preparation with 10⫺4 M ACh (Ca2⫹
measurement data sets) or 10⫺3 M SNP (all other data sets)
was recorded for each observed arteriole and is reported as
the maximum.
Agonist Application
Pressurized glass micropipettes placed at the vessel wall
were used for localized agonist application [ATP, Ado, AMP,
ACh, or norepinephrine (NE)] as previously described (28).
Flow out of the pipette (⬃10 ␮m tip diameter) was achieved
by a manometer system (30 cmH2O ejection pressure). FITCdextran (100 ␮M) or 2% Texas red-dextran was added to the
contents of the pipette, and brief epi-illumination was used to
confirm that flow out of the pipette was over the local site
(⬃100 ␮m length of vessel on either side of the pipette) only
and to verify that the superfusion solution carried the pipette
contents away from the upstream site. From the diffusion
constants for small molecules (between 10⫺6 and 10⫺5 cm2/s)
such as those applied in this study and the distance between
the local application and upstream observation site (⬎1,000
␮m), it is apparent that diffusion is insufficient to account for
Fig. 1. Schematic (not to scale) of the experimental observation site consisting of a third-order arteriole originating
from a feed arteriole. Arrows in the lumen indicate the direction of blood flow. Local and upstream observation sites
are shown. Gray arrow at the local site indicates the direction of flow of micropipette contents being washed away
from the upstream site by the flowing superfusion solution. A cannulating micropipette is positioned in another
third-order arteriole arising from the same parent vessel as the test arteriole. Occluding rod A is temporarily
placed to inhibit blood flow and allow perfusion of the cannulating micropipette contents through the vascular
network. During BAPTA loading only, occluding rod B is placed at the position indicated to limit BAPTA loading
to the upstream half of the test vessel. Blood remains in the vascular network downstream of this temporary
occluding rod.
AJP-Heart Circ Physiol • VOL
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accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council).
Adult male Golden hamsters (110–160 g body wt) were
anesthetized with an intraperitoneal injection of pentobarbital sodium (70 mg/kg) and tracheotomized to maintain a
patent airway. A femoral venous catheter was placed for
administration of supplemental pentobarbital sodium as
needed during surgery and for constant infusion (10 mg/ml at
0.56 ml/h) throughout the experimental protocol. The depth
of anesthesia was assessed by monitoring the hamster’s reflex withdrawal to a toe pinch. A femoral arterial catheter
was placed to monitor the animal’s mean arterial blood
pressure (⬃100 mmHg). Hamster body temperature was
maintained at 37°C via convective heat. The left cheek pouch
was exteriorized and prepared for in situ intravital microscopic observations as described previously (14). Briefly, the
left cheek pouch was everted, cut longitudinally, and gently
spread over a semicircular lucite pedestal using insect pins,
and excess connective tissue was carefully cleared. During
surgery and experimental protocols, the cheek pouch preparation was continuously superfused (at ⬃5 ml/min) with a
bicarbonate-buffered physiological salt solution warmed to
36°C containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2
MgSO4, and 30.0 NaHCO3 and equilibrated with 5% CO295% N2 to maintain pH 7.40 ⫾ 0.05. At the completion of all
experimental protocols, animals were administered a lethal
dose of pentobarbital sodium.
After surgery, the preparation was allowed to stabilize for
45–60 min before data collection. Third-order arterioles
(maximum diameter ⬃40 ␮m) located in the central region of
the cheek pouch preparation were chosen for study and
visualized using an Olympus BX50WI microscope. Unless
otherwise specified, the tissue was transilluminated with a
tungsten lamp, and the arteriole was imaged using a ⫻25
objective (numerical aperture 0.35), displayed on a Sony
monitor using a CCD camera (Dage MTI CCD72S), and
recorded on videotape. Vessel diameter was measured offline
using video calipers generated by a modified video analyzer
(model 321, Colorado Video), which was calibrated with a
videotaped stage micrometer. Observations were made dur-
H27
H28
ARTERIOLAR DILATIONS INITIATED BY PURINES
of interest of dye-loaded endothelium (from digitized images
using Scion Image software). The EC Ca2⫹ change is expressed as the relative change in fluorescence intensity ratio
from baseline. Diameter measurements were made from the
same digitized images using a calibrated stage micrometer
and expressed as a percentage of normalized baseline diameter.
Endothelium Denudation With Air Embolism
EC Ca2⫹ Chelation
Cannulating micropipettes were triple beveled to create a
sharp tip (diameter 7–10 ␮m) to facilitate penetration
through connective tissue and the arteriolar wall. To selectively remove ECs, an arteriole was cannulated with a pipette containing air. Once the pipette entered the vessel
lumen, it was rapidly pressurized to perfuse a local region of
the microvascular network with air, which could be verified
visually as the vessel was completely cleared of blood by the
flow of air. The pipette was depressurized immediately and
removed. Within 5–10 min the bubble would disperse into
the microvasculature, and blood flow would resume. Air bubbles were rarely seen in venules. Air treatment generally
denuded a limited length of vessel (⬃200 ␮m). A period of
20–25 min was allowed for vessel tone to reestablish before
data collection. Selective disruption of ECs could be verified
visually as SMCs were slightly constricted and occasional
platelet interactions with the vessel wall could be seen. The
following functional response was used as the identifying
criterion for an arteriolar region with selective endothelium
disruption: lack of dilator response to ACh despite the preservation of intact smooth muscle functionality defined as it’s
ability to contract (with ATP) and relax (with SNP).
To selectively buffer the EC Ca2⫹, the microvascular network was intraluminally perfused with 5 ␮M BAPTA (AM) as
described for fura-PE3 (AM). An additional occluding rod
(Fig. 1, temporary occluding rod B) was placed between the
upstream and downstream sites of the test arteriole to confine BAPTA loading to half the vessel length (⫹BAPTA).
Blood remained in regions downstream of this occluding rod
(⫺BAPTA). After 15 min of BAPTA (AM) perfusion, the
pipette and occluding rods were removed, and blood flow was
allowed to resume. Twenty minutes was allowed for intracellular deesterification of the molecule. The following functional response was used as the criterion for selective chelation of EC Ca2⫹: inhibition of dilator response to ACh despite
preservation of intact smooth muscle functionality defined as
it’s ability to contract (with NE) and relax (with SNP).
EC Ca2⫹ Measurement
EC Ca2⫹ measurements were made by using the indicator
fura-PE3 as previously described (25). Briefly, after identification of the test arteriole, another third-order arteriole
arising from the same parent vessel was cannulated with a
triple-beveled micropipette containing 2 ␮M fura-PE3 (AM)
dye solution (Fig. 1). Once the pipette had entered the vessel
lumen, it was pressurized to intraluminaly perfuse the microvascular network. Areas of perfusion could be verified
visually as blood was completely cleared from the vessel by
flow of the dye. Up to two blunted, curved glass-occluding
rods were gently placed in upstream or downstream regions
to temporarily inhibit blood flow and direct flow of fura-PE3
(AM) down the test arteriole. After 30 min of dye perfusion
(total volume ⬃20 ␮l), the pipette and occluding rods were
removed and blood flow was allowed to resume. Thirty minutes were allowed for intracellular deesterification of the dye
and reestablishment of vessel tone before data collection. In
a previous study (26), selective dye loading of ECs was
confirmed using functional criteria.
Dye-loaded arterioles were visualized using a ⫻40 long
working distance water immersion objective (Olympus, numerical aperture 0.8). Fura-PE3 was excited using a 100-W
mercury arc lamp and either 340 ⫾ 8 or 380 ⫾ 7 nm narrow
bandpass filters using an optical switch (DX-1000, Solamere
Technology Group) with a 140-ms flash at each wavelength
at 1 Hz. Emissions at 510 ⫾ 40 nm were imaged via a linear
eight-bit ICCD camera (XR GEN III ICCD, Stanford Photonics) and captured with a Scion CG-7 data acquisition board
and Scion Image (version 1.62c) software on a Macintosh G3
computer.
EC Ca2⫹ was estimated offline as the ratio of fluorescence
emissions intensity (background subtracted) of fura-PE3 at
excitation wavelengths of 340 and 380 nm in defined regions
AJP-Heart Circ Physiol • VOL
Protocols
ATP and Ado dose response. Concentrations of 10⫺7
M–10⫺3 M ATP or 10⫺7 M–10⫺4 M Ado were micropipette
applied as described above to determine the dose response of
arterioles to these purines. Observations were first made at
the local site. After a brief recovery period (⬃10 min), observations were made upstream. In preliminary experiments it
was verified that multiple applications and the order of
observation (local followed by upstream or vice versa) did not
affect the arteriole’s response. Paired local and remote observations were made on the same vessel in all experiments
unless specified otherwise. Only one concentration of each
agonist was usually tested on each arteriole.
Role for P2x, P2, and P1 receptors in ATP-initiated response.
The P2 receptor family is divided into two subtypes, P2X and
P2Y. P2X receptors are ATP-gated cation channels and allow
direct entry of Na⫹ and Ca2⫹. P2Y receptors are coupled to G
proteins and initiate phospholipase-based signal transduction via mobilization of inositol trisphosphate-sensitive Ca2⫹
stores (1, 13). To identify the purinergic receptor being activated by extracellular ATP, the local and upstream vasomotor response to 10⫺4 M ATP was first recorded as described
above (control data). The entire cheek pouch preparation was
then exposed (in separate experiments) to either 1) 10⫺5 M
pyridoxal-phosphate-6-azophenyl-2⬘,4⬘-disulfonic acid tetrasodium (PPADS, P2X antagonist); 2) 10⫺5 M suramin (P2X
and P2Y antagonist); or 3) 10⫺6 M xanthine amine congener
(XAC, P1 antagonist) by adding the agent to the superfusion
solution. After 30 min of exposure to the antagonist, the same
vessel’s response to ATP was recorded again in the continued
presence of the blocker.
Role for hydrolysis of ATP to Ado during the response to
ATP application. To determine whether hydrolysis of ATP to
Ado plays a role in the dilator response to ATP, observations
were first made at the local and upstream sites during 10⫺4
M ATP application and at the local site only during 10⫺4 M
AMP and 10⫺4 M Ado (in separate arterioles) exposure in the
same preparation (control data). The entire tissue preparation was then exposed to 10⫺4 M ␣,␤-methylene adenosine
5⬘-diphosphate (AOPCP, 5⬘-ectonucleotidase inhibitor) by
adding it to the superfusion solution. After 30 min of expo-
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the conducted response (2, 26). In addition, it has been shown
that not all agonists that produce large local dilations (e.g.,
SNP) initiate upstream responses even when applied for 2
min (26). This provides functional evidence that diffusion or
convection of local agonists do not underlie upstream responses. Fluorescent tracers themselves do not affect arteriolar responses (17).
ARTERIOLAR DILATIONS INITIATED BY PURINES
Materials
A 20-␮l aliquot of 10⫺3 M fura-PE3 (AM) (TEF Labs,
Austin, TX; dissolved in 100% DMSO) and 4 ␮l of 12.5 mg/ml
Pluronic-127 (TEF Labs, Austin, TX; made in 100% DMSO)
stock solutions were mixed and diluted in 10 ml 0.9% NaCl
(dye solution). This resulted in a final concentration of 2 ␮M
fura-PE3 (AM), 5 ␮g/ml Pluronic-127, and 2.4 ␮l/ml DMSO. A
50-␮l aliquot of 10⫺3 M BAPTA (AM) (Molecular Probes,
Eugene, OR; dissolved in 100% DMSO) and 20 ␮l of 12.5
mg/ml Pluronic-127 (TEF Labs; made in 100% DMSO) stock
solutions were mixed and diluted in 10 ml 0.9% NaCl (Ca2⫹
buffer solution). This resulted in a final concentration of 5
␮M BAPTA (AM), 25 ␮g/ml Pluronic-127, and 7.0 ␮l/ml
DMSO.
AJP-Heart Circ Physiol • VOL
All other reagents were obtained from Sigma (St. Louis,
MO). Solutions were prepared fresh daily in superfusion
solution.
Data Analysis and Statistics
Typically, only one arteriole was studied in each animal. In
some experiments (e.g., dose response, AOPCP), up to three
arterioles were observed, but a different agonist and/or concentration was tested in each case to avoid introduction of
bias for a particular animal in the averaged data set for any
given condition. The number of observations (n) refers to the
number of arterioles studied. All data are reported as
means ⫾ SE. Data are expressed normalized to baseline
(340-to-380-nm ratio and percent diameter) or as an absolute
diameter change (in ␮m) over 10-s intervals relative to baseline (averaged over 1 min). Responses from multiple experiments were analyzed by repeated-measures ANOVA with
Dunnett’s multiple-comparison posttest or paired Student’s
t-test as appropriate to determine statistical differences compared with baseline. Changes were considered significant if
P ⬍ 0.05.
RESULTS
The number of arterioles studied, resting and maximum vessel diameters, and local to upstream site distance for all experiment sets are summarized in Table
1. The time at which the peak response occurred varied
between vessels (by up to ⬃30 s). In the figures, the
averaged time course refers to the mean observation at
each time point for multiple arterioles, whereas peak
response refers to the mean of the peak response from
multiple arterioles. For the sake of clarity, data in the
text refer to the mean peak response.
Dose Response to ATP and Ado
Application of 10⫺7 M to 10⫺3 M ATP caused a
dose-dependent constriction (Fig. 2A) followed by dilation locally (Fig. 2B) and dilation (but no constriction)
upstream (Fig. 2C). The peaks occurred 40 ⫾ 10, 130 ⫾
10, and 110 ⫾ 10 s after the onset of ATP application
for the local constriction, local dilation, and upstream
dilation, respectively. Peak constriction was observed
at 10⫺4 M ATP. The magnitude of the local and upstream dilator responses to ATP were identical (Fig. 2,
B vs. C, filled squares, P ⬎ 0.05). Application of 10⫺7 M
to 10⫺4 M Ado caused a dose-dependent dilation, but
not constriction, both locally (Fig. 2B) and upstream
(Fig. 2C). The times at which the peak dilations occurred were 70 ⫾ 10 and 110 ⫾ 10 s after the onset of
Ado application for the local and upstream response,
respectively. On average, the local dilator responses to
Ado were slightly larger than the upstream responses
(Fig. 2, B vs. C, open squares, P ⬍ 0.05). However, the
local and upstream dilator responses between ATP and
Ado were indistinguishable (P ⬎ 0.05). All subsequent
experiments were conducted using 10⫺4 M ATP or Ado,
because this concentration of ATP clearly stimulated
P2 receptors (indicated by constriction) without saturating the vessel’s dilator capacity.
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sure to the antagonist, the responses to ATP, AMP, and Ado
were recorded again in the continued presence of the blocker.
Identification of the vascular cell type initiating the response to ATP and Ado. To identify the primary cell type
(endothelial vs. smooth muscle) responsible for initiation of
the vasomotor responses to ATP and Ado, observations were
made before and after selective removal of ECs via air embolism in the same preparation. Observations were first
made in intact arterioles with local application of 10⫺4 M
ACh, 10⫺4 M ATP, or 10⫺4 M Ado (control data), following
which ECs were removed as described above. The arteriolar
responses of the endothelium-denuded region to local application of ACh, ATP, and Ado were then recorded. Subsequent
to collection of all local agonist application data in each
animal, the entire preparation was exposed to 10⫺3 M SNP
by adding it to the superfusion solution, and the vasomotor
response of the endothelium-removed region was recorded.
EC Ca2⫹ response to ATP and Ado. To test whether the
arteriolar dilation associated with ATP or Ado stimulation
involves EC Ca2⫹ as a signaling intermediate, we measured
local and upstream changes in EC Ca2⫹ and vessel diameter
initiated by 10⫺4 M ATP or 10⫺4 M Ado. Paired local and
remote observations were not always made on the same
vessel in these experiments, because we were limited to
collecting data in arteriolar regions that were fura-PE3
loaded (for Ca2⫹ measurement) and in focus (for vessel diameter measurement). Subsequent to collection of all micropipette agonist application data in each animal, the entire
cheek pouch preparation was exposed to 10⫺4 M ACh (an
agonist known to maximally increase intracellular Ca2⫹ in
the endothelium) and EC Ca2⫹ was measured.
Role of change in EC Ca2⫹ in the response to P1 receptor
stimulation. To determine whether the change in EC Ca2⫹
associated with arteriolar dilation to purines is a required
signaling intermediate, observations were made in the same
arterioles before and after chelation of EC Ca2⫹. Local and
upstream responses to 10⫺4 M Ado and the local response to
10⫺4 M ACh were first recorded under control conditions.
After BAPTA was loaded, local responses to Ado, ACh, 10⫺4
M NE, and 10⫺3 M SNP applied to the BAPTA-perfused site
(⫹BAPTA) were observed. Ado was also applied to the downstream site that remained blood filled and observations were
made at this local site (⫺BAPTA) and at a BAPTA-loaded
upstream site. Regions of the vasculature that were BAPTA
perfused did not completely regain spontaneous tone; hence,
5 min before the beginning of data collection, the superfusion
solution was changed to one containing 10⫺7 M NE to augment tone. This was done for both control and BAPTA-loaded
conditions to ensure that NE itself does not affect local and
upstream responses.
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ARTERIOLAR DILATIONS INITIATED BY PURINES
Table 1 Number of arterioles observed, baseline and maximum diameters, and local-to-upstream site distance
for all experiment sets
Data Set
Local
Baseline
Upstream
Baseline
Maximum
Diameter
Local Upstream
Distance
6–8*
6*
21.8 ⫾ 1.1
16.8 ⫾ 1.2
22.2 ⫾ 1.1
16.4 ⫾ 1.2
37.7 ⫾ 1.7
32.0 ⫾ 2.1
1,260 ⫾ 30
1,210 ⫾ 30
8
22.4 ⫾ 2.1
22.9 ⫾ 2.1
22.4 ⫾ 1.9
22.7 ⫾ 1.5
34.5 ⫾ 2.6
1,200 ⫾ 50
8
24.4 ⫾ 1.8
26.7 ⫾ 1.8
23.2 ⫾ 1.4
27.7 ⫾ 2.1
40.2 ⫾ 1.6
1,110 ⫾ 40
7
20.6 ⫾ 2.7
22.4 ⫾ 2.5
20.1 ⫾ 2.6
21.2 ⫾ 2.4
35.5 ⫾ 4.0
1,290 ⫾ 90
6
25.8 ⫾ 2.3
27.6 ⫾ 1.9
25.8 ⫾ 2.8
28.3 ⫾ 2.8
41.7 ⫾ 2.3
1,290 ⫾ 90
6
31.5 ⫾ 0.3
24.0 ⫾ 0.3
10–12
8
21.4 ⫾ 1.8
21.2 ⫾ 1.9
22.5 ⫾ 1.4
21.9 ⫾ 2.1
4
17.3 ⫾ 0.9
26.5 ⫾ 2.0
17.7 ⫾ 3.9
16.2 ⫾ 2.4
27.2 ⫾ 5.3
44.8 ⫾ 1.2
1,140 ⫾ 40
1,090 ⫾ 30
37.1 ⫾ 5.5
1,090 ⫾ 40
Values are means ⫾ SE in ␮m; n, number of arterioles studied. * n at each concentration. ⫺EC, endothelium-denuded region; ⫹BAPTA,
region of arteriole that was BAPTA loaded; ⫺BAPTA, region of arteriole that was not BAPTA loaded; Ado, adenosine; PPADS, pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid tetrasodium; XAC, xanthine amine congener; AOPCP, ␣,␤-methylene adenosine 5⬘-diphosphate.
ATP-Induced Constrictions are Via P2X Receptors and
Dilations are Via P1 Receptors
dilations (11.6 ⫾ 1.2 vs. 3.0 ⫾ 1.0 ␮m, P ⬍ 0.05) were
inhibited (Fig. 5C).
To identify the role of P2X receptors in the response
to ATP, the local (Fig. 3A) and upstream (Fig. 3B)
vasomotor responses were observed before (control)
and during treatment with PPADS, a selective P2X
antagonist. In the presence of PPADS, the local constriction to ATP was abolished (⫺12.4 ⫾ 3.4 vs. ⫺0.2 ⫾
0.2 ␮m, P ⬍ 0.05; control vs. treatment), whereas the
local (8.4 ⫾ 1.3 vs. 8.4 ⫾ 1.0 ␮m, P ⬎ 0.05) and
upstream dilations (7.2 ⫾ 1.6 vs. 7.0 ⫾ 1.4 ␮m, P ⬎
0.05) were unaffected (Fig. 3C).
To determine the role of P2 receptors in the local
(Fig. 4A) and upstream (Fig. 4B) responses to ATP, the
nonselective P2 antagonist suramin was used. Exposure to suramin caused a slight increase in resting
arteriolar diameter, although the vessels clearly retained their capacity to dilate (Table 1). In the presence
of suramin, the local constriction to ATP was attenuated (⫺9.6 ⫾ 1.9 vs. ⫺3.5 ⫾ 1.7 ␮m, P ⬍ 0.05), whereas
the local (7.1 ⫾ 1.4 vs. 10.3 ⫾ 0.9 ␮m, P ⬎ 0.05) and
upstream (5.7 ⫾ 1.1 vs. 7.2 ⫾ 1.3 ␮m, P ⬎ 0.05)
dilations were not (Fig. 4C).
To investigate whether the local (Fig. 5A) and upstream (Fig. 5B) dilator response to ATP is produced
via stimulation of P1 receptors, a nonselective P1 antagonist XAC was used. With XAC, the magnitude of
the local constriction to ATP was unchanged (⫺9.5 ⫾
1.7 vs. ⫺10.2 ⫾ 2.1 ␮m, P ⬎ 0.05), whereas the local
(7.9 ⫾ 1.8 vs. 2.5 ⫾ 1.1 ␮m, P ⬍ 0.05) and upstream
Hydrolysis to Ado Facilitates ATP-Induced Dilation
AJP-Heart Circ Physiol • VOL
Nucleotidases present on the cell’s extracellular
membrane degrade adenine nucleotides to Ado. 5⬘Ectonucleotidase is the enzyme implicated in the final
step of Ado formation via this pathway, catalyzing the
breakdown of AMP to Ado. To test whether ATP hydrolysis to Ado, with subsequent stimulation of P1
receptors (vs. direct binding of ATP to P1 receptors),
was the trigger for the dilation, we used AOPCP, which
is a specific inhibitor of 5⬘-ectonucleotidase (Fig. 6).
Treatment with AOPCP caused a slight increase in
resting diameter, although the vessels still retained
their capacity to dilate (Table 1). AOPCP attenuated
the local dilation to AMP (16.0 ⫾ 1.7 vs. 6.1 ⫾ 1.4 ␮m,
P ⬍ 0.05) but not Ado (16.1 ⫾ 3.2 vs. 14.7 ⫾ 2.3 ␮m, P ⬎
0.05). In the presence of AOPCP, the local constriction
to ATP was unchanged (⫺8.8 ⫾ 2.5 vs. ⫺9.6 ⫾ 1.8 ␮m,
P ⬎ 0.05). The local (12.4 ⫾ 1.6 vs. 5.3 ⫾ 1.9 ␮m, P ⬍
0.05) and upstream (9.6 ⫾ 1.6 vs. 7.0 ⫾ 1.7 ␮m, P ⬍
0.05) dilations to ATP were attenuated in the presence
of AOPCP.
Initiation of Dilator Signals by Purines Occurs
Primarily in ECs
To determine whether ECs or SMCs are primarily
responsible for the initiation of vasomotor responses to
purines, observations were made during agonist appli-
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Dose response
ATP
Ado
PPADS
Control
Blocker
Suramin
Control
Blocker
XAC
Control
Blocker
AOPCP
Control
Blocker
EC denudation
Control
⫺EC
Fura-PE3
ATP
Ado
BAPTA
Control
⫹BAPTA
⫺BAPTA
n
ARTERIOLAR DILATIONS INITIATED BY PURINES
H31
their capacity to dilate to SNP (20.5 ⫾ 3.9 ␮m, P ⬍ 0.05
from baseline).
EC Ca2⫹ Increases in Response to ATP and Ado
ATP application increased EC Ca2⫹ at both the local
(Fig. 8A) and upstream (Fig. 8B) site. The increase in
the average peak fura ratio was 33 ⫾ 5% locally and
19 ⫾ 3% upstream (P ⬍ 0.05 from baseline). The
change in diameter was the same as that described
earlier, i.e., a biphasic response at the local site and
only dilation upstream. Ado application also caused an
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Fig. 2. Dose response of arteriolar diameter change in response to 2
min of ATP or adenosine (Ado). Values are means ⫾ SE (n ⫽ 6 to 8).
A: peak local constrictions to ATP. B: peak local dilations to ATP and
Ado. C: peak upstream dilations to ATP and Ado. See text for
statistical analysis results.
cation at the local site in vessels with intact ECs (Fig.
7A) or following selective removal of ECs (Fig. 7B). Air
embolism resulted in an increase in resting arteriolar
tone (Table 1). The average peak responses are shown
in Fig. 7C. EC denudation abolished ACh-induced dilations (17.9 ⫾ 2.1 vs. 1.6 ⫾ 0.9 ␮m, P ⬍ 0.05), establishing that ECs were successfully disrupted with air
treatment. In EC-denuded vessels, the magnitude of
the local constriction to ATP was unchanged (⫺18.8 ⫾
2.5 vs. ⫺21.4 ⫾ 4.8 ␮m, P ⬎ 0.05), whereas the dilation
(8.5 ⫾ 2.2 vs. 0.6 ⫾ 0.4 ␮m, P ⬍ 0.05) was abolished.
The dilation to Ado was significantly reduced in ECdenuded vessels (19.2 ⫾ 2.3 vs. 4.1 ⫾ 0.9 ␮m, P ⬍ 0.05).
As expected, endothelium-denuded vessels maintained
AJP-Heart Circ Physiol • VOL
Fig. 3. Response of arterioles to 2 min of ATP (10⫺4 M) application in
the absence (control) or presence of the P2X receptor antagonist
pyridoxal-phosphate-6-azophenyl-2⬘,4⬘-disulfonic acid tetrasodium
(PPADS, 10⫺5 M). Values are means ⫾ SE (n ⫽ 8). A: averaged time
course of the local change in diameter. B: averaged time course of the
upstream change in diameter. C: peak local and upstream changes in
diameter. * Significantly different from control response (P ⬍ 0.05).
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ARTERIOLAR DILATIONS INITIATED BY PURINES
Dilation by P1 Receptor Stimulation Requires an
Increase in EC Ca2⫹
To assess whether an increase in EC Ca2⫹ is required for the arteriolar dilations associated with purinergic stimulation, observations were made in con-
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Fig. 4. Response of arterioles to 2 min of ATP (10⫺4 M) application in
the absence (control) or presence of the P2 receptor antagonist
suramin (10⫺5 M). Values are means ⫾ SE (n ⫽ 8). A: averaged time
course of the local change in diameter. B: averaged time course of the
upstream change in diameter. C: peak local and upstream changes in
diameter. * Significantly different from control response (P ⬍ 0.05).
increase in EC Ca2⫹ at both the local (Fig. 8C) and
upstream (Fig. 8D) site. In this case, the increase in the
average peak fura ratio was 29 ⫾ 4% locally and 17 ⫾
6% upstream (P ⬍ 0.05 from baseline). Again, as described earlier, the vasomotor response to Ado involved
only dilations. With ACh, the increase in the average
peak fura ratio was 168 ⫾ 32% (P ⬍ 0.05 from baseline
and all purine responses). This confirmed that our
system had the capacity to detect changes in EC Ca2⫹
greater than those recorded during purinergic stimulation should they have occurred.
AJP-Heart Circ Physiol • VOL
Fig. 5. Response of arterioles to 2 min of ATP (10⫺4 M) application in
the absence (control) or presence of the P1 receptor antagonist xanthine amine congener (XAC, 10⫺6 M). Values are means ⫾ SE (n ⫽
7). A: averaged time course of the local change in diameter. B:
averaged time course of the upstream change in diameter. C: peak
local and upstream changes in diameter. * Significantly different
from control response (P ⬍ 0.05).
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ARTERIOLAR DILATIONS INITIATED BY PURINES
H33
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Fig. 6. Peak diameter change of arterioles in the absence (control) or
presence of the 5⬘-ectonucleotidase inhibitor ␣,␤-methyleneadenosine 5⬘-diphosphate (AOPCP, 10⫺4 M) in response to AMP (10⫺4
M, local, n ⫽ 6), Ado (10⫺4 M, local, n ⫽ 5), and ATP (10⫺4 M, local
and upstream, n ⫽ 6). Values are means ⫾ SE. * Significantly
different from control response (P ⬍ 0.05).
trol vessels and following buffering of EC Ca2⫹ with
the Ca2⫹ chelator BAPTA. Loading of BAPTA resulted
in a decrease in resting arteriolar tone. However, the
vessels retained their capacity to dilate (Table 1). In
the ⫹BAPTA region of the arteriole, ACh-induced dilation was significantly reduced (16.3 ⫾ 3.9 vs. 4.6 ⫾
2.4 ␮m, P ⬍ 0.05), establishing successful buffering of
EC Ca2⫹ (Fig. 9A). The local response to Ado (Fig. 9B)
was abolished in the ⫹BAPTA region (15.2 ⫾ 3.0 vs.
2.2 ⫾ 1.1 ␮m, P ⬍ 0.05), whereas in the ⫺BAPTA
region it remained intact (12.4 ⫾ 2.4 ␮m, P ⬎ 0.05
compared with control). Buffering EC Ca2⫹ also abolished the manifestation of the upstream dilation (8.7 ⫾
1.3 vs. 2.4 ⫾ 1.1 ␮m, P ⬍ 0.05, Fig. 9C) despite its
initiation at the local site. BAPTA-loaded regions constricted to NE (⫺24.5 ⫾ 4.2 ␮m, P ⬍ 0.05 from baseline), indicating selective buffering of EC Ca2⫹, and
dilated to SNP (14.5 ⫾ 4.3 ␮m, P ⬍ 0.05 from baseline),
indicating ample dilator capacity (Fig. 9D).
DISCUSSION
The present study demonstrates that in intact bloodperfused arterioles, stimulation of P1 and not P2 purinergic receptors initiates a vasodilator response, which
spreads axially along the vessel wall to upstream regions. There is a corresponding increase in local EC
Ca2⫹ that is also conducted upstream. Local and upstream increases in EC Ca2⫹ are required for manifestation of the respective responses. Furthermore, we
show that intact endothelium is required for the initiation of the dilator signal. In contrast to the dilation,
constriction initiated by purines is a result of stimulation of P2X purinergic receptors. This constriction remains localized. It is independent of endothelium and
occurs via direct stimulation of receptors on SMCs.
ATP produces a dose-dependent constriction followed by dilation at the site of receptor occupation.
Only the dilator signal is propagated along the vessel
wall and results in an upstream vasomotor response.
Ado causes dose-dependent dilations at the site of
receptor stimulation and, like ATP, dilation upstream.
AJP-Heart Circ Physiol • VOL
Fig. 7. Averaged time course of local change in diameter in response
to 2 min of ATP (10⫺4 M), Ado (10⫺4 M), and ACh (10⫺4 M) application in control arterioles with intact endothelium (A, n ⫽ 6) or
endothelium-denuded arterioles (B, n ⫽ 7). Values are means ⫾ SE.
Smooth muscle cell functionality of endothelium-denuded arterioles
(B) is demonstrated by their ability to constrict (with 10⫺4 M ATP)
and dilate (with 10⫺3 M SNP). C: peak changes in diameter of control
(⫹EC) and endothelium-denuded (⫺EC) arterioles. * Significantly
different from control response (P ⬍ 0.05).
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ARTERIOLAR DILATIONS INITIATED BY PURINES
The magnitude of the dilator responses to each of these
purines is the same, which, based on their dose-response curves, is consistent with both purines acting
via the same signaling pathway. Constrictions to Ado
[reported by Doyle et al. (12) following stimulation of
the A3 receptor subtype in isolated arterioles] were
never observed in the in situ vascular preparation of
the current study.
Inhibition of P2X receptors with PPADS abolished
the local constriction without affecting the local and
remote dilations. Consistent with interpretations of
studies conducted on isolated vessels (6, 23), our results show that ATP causes constriction via P2X receptors. Additionally, our results show that the dilator
response is not linked to the P2X receptor pathway.
These findings with PPADS strongly support that at
the receptor level the local constriction and dilation do
not originate at the same source but rather the biphasic response to extracellular ATP is the result of activation of at least two independent pathways.
Inhibition of P2 receptors with suramin attenuated
the local constriction to ATP (confirming effective inAJP-Heart Circ Physiol • VOL
hibition of P2 receptors at the concentration used in our
study) but surprisingly did not reduce the dilator responses. In several systems, the vasodilator action of
ATP has been associated with stimulation of P2Y receptors, but in many cases this link lacks explicit
confirmation (13, 35). Our findings show that in an
intact system, P2 receptors are not involved in the local
and upstream dilations initiated by ATP. We speculate
that because the architecture of the vascular and parenchymal cells is intact, highly active nucleotidases
present in the extracellular space (discussed below)
modulate the ATP response in ways that are distinct
from cells in culture.
The finding that the ATP induced local and propagated dilations do not involve P2 receptors led us to the
hypothesis that ATP was acting via stimulation of a P1
receptor pathway. Consistent with this hypothesis, direct stimulation of P1 receptors with Ado produces local
and upstream dilations that are not different from
those initiated by ATP (dose response and EC Ca2⫹
data). In fact, blocking P1 receptors with XAC resulted
in a complete inhibition of the dilations to ATP,
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Fig. 8. Averaged time course of diameter (filled symbols, right y-axis) and EC Ca2⫹ (open symbols, left y-axis) in
response to ATP (10⫺4 M) or Ado (10⫺4 M) application in fura-PE3-loaded arterioles. Values are means ⫾ SE.
Diameter measurements are expressed as percent change from normalized baseline conditions. EC Ca2⫹ changes
are expressed as relative change (from normalized baseline conditions) in the ratio of fluorescence emissions
intensity of fura-PE3 at excitation wavelengths of 340 nm and 380 nm. A: ATP local (n ⫽ 12); B: ATP upstream (n ⫽
10); C: Ado local (n ⫽ 8); D: Ado upstream (n ⫽ 8).
ARTERIOLAR DILATIONS INITIATED BY PURINES
H35
whereas the constriction was unaffected. This further
supports that the constriction and dilation are mediated by independent pathways and demonstrates that
P1 receptors play a crucial role in the local and upstream dilator response to ATP. A similar outcome was
obtained using a different P1 receptor antagonist,
8-phenyltheophylline (n ⫽ 3, data not shown). Little is
known about the distribution of P1 receptor subtypes
(A1, A2a, A2b, and A3) in microvessels, which is why
XAC, an antagonist that is nonselective between subtypes of P1 receptors, was used at a concentration
known to inhibit dilations to Ado in small arterioles
(27). Identification of the specific P1 receptor involved
in the dilator responses to ATP was beyond the scope of
the current study.
There are two obvious mechanisms by which ATP
could be stimulating P1 receptors. First, even though
ATP preferentially binds P2 over P1 receptors, at the
AJP-Heart Circ Physiol • VOL
concentrations used in this study ATP itself could be
binding P1 receptors. Alternatively, ectoenzymes could
be degrading ATP to Ado, which in turn stimulates P1
receptors. To distinguish between these two possibilities, AOPCP, a specific inhibitor of 5⬘-ectonucleotidase,
was used to block the final step of the conversion of
ATP to Ado. This protein presumably represents the
major enzyme responsible for the formation of extracellular nucleoside from nucleoside 5⬘-monophosphates
and thus plays an important role in the formation of
Ado from AMP (34). However, it should be kept in mind
that the pattern of catalytic activities at the cell surface is in actuality much more complicated than the
linear hydrolysis chain from ATP to Ado assumed here,
and any product of each hydrolysis step is itself likely
to contribute to triggering a vasomotor response. As
expected, AOPCP dramatically decreased the local dilation to AMP without affecting that to Ado, verifying
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Fig. 9. A: averaged time course of the local response to 2 min of ACh (10⫺4 M) in control and applied to the
BAPTA-perfused region (⫹BAPTA). B: averaged time course of the local response to 2 min of Ado (10⫺4 M) in
control and applied to the BAPTA-perfused region (⫹BAPTA) and the region that remained blood filled during
BAPTA loading (⫺BAPTA). C: averaged time course of the upstream change in diameter in control and in the
BAPTA-perfused region (⫹BAPTA) in response to 2 min of Ado application. D: peak changes in diameter of control,
BAPTA-loaded region (⫹BAPTA), and region remaining blood filled during BAPTA loading (⫺BAPTA) in response
to ACh, Ado, norepinephrine (NE, 10⫺4 M), and sodium nitroprusside (SNP, 10⫺3 M). Values are means ⫾ SE (n ⫽
4). * Significantly different from control response (P ⬍ 0.05).
H36
ARTERIOLAR DILATIONS INITIATED BY PURINES
AJP-Heart Circ Physiol • VOL
Furthermore, influx of Ca2⫹ into ECs as a consequence
of membrane hyperpolarization has been shown (21).
Our findings thus support a model in which the conduction of a hyperpolarizing signal transmitted axially
via the endothelium acts as the trigger for the upstream increase in EC Ca2⫹ and subsequent dilation.
An alternative hypothesis is that the local increase in
EC Ca2⫹ caused by P1 receptor stimulation spreads
from cell to cell by gap junction channels or via a
paracrine pathway as has been established in other
systems (8, 33) and underlies the resulting propagated
dilations. The time course of the change in EC Ca2⫹
and associated dilations that we observed suggests
that the increase in Ca2⫹ is related to the initiation of
a vasodilator signal while the maintenance of the dilation is achieved by other means. Such mechanisms
could involve changes in Ca2⫹ sensitivity, as demonstrated in SMCs (4), or be independent of changes in
endothelial whole cell Ca2⫹ (24). Whether the rise in
EC Ca2⫹ triggers the release of an endothelium-derived dilator or hyperpolarizes the EC (e.g., via activation of Ca2⫹-dependent K⫹ channels), either of which
could subsequently act on SMCs via paracrine or
myoendothelial mechanisms, is unknown.
In conclusion, it is demonstrated here that stimulation of P1 receptors on ECs produces an increase in EC
Ca2⫹ as well as a decrease in vascular resistance that
spreads axially along the vessel wall to remote regions.
Collectively, these findings expand the current understanding (9, 10, 19, 22) of how dilator responses to ATP
may be initiated and transmitted in an intact bloodperfused system in situ. In addition, our novel identification of the involvement of EC Ca2⫹ in the conducted
response reveals EC Ca2⫹ signaling as a required signaling intermediate for the manifestation of communicated vasodilator signals throughout the microvasculature. Thus the current study advances the understanding of signaling pathways by which blood flow is
regulated, particularly during the complex integrated
response of arterioles to local metabolites.
We thank Coral L. Murrant for contributions to this work and
Patricia A. Titus for skilled technical assistance.
This study was supported by National Heart, Lung, and Blood
Institute Grant RO1-HL-56574.
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